Ultracentrifuge

Figure 1: Modern ultracentrifuge

Figure 2: Modern benchtop ultracentrifuge

The ultracentrifuge is a centrifuge optimized for high speeds . Modern ultracentrifuges (UZ) can achieve accelerations of up to 10 ⁶ g and speeds of up to 150,000 revolutions per minute.

The UZ is an extremely important laboratory instrument and an indispensable part of many areas of scientific research. In cell biology , molecular biology and microbiology it is used for fractionation and purification of cellular components, in nanotechnology for the purification and separation of nanoparticles .

A general distinction is made between two types of ultracentrifuges, the preparative and the analytical .

The areas of application of the preparative UZ include above all the pelleting and purification of fine particle fractions, such as cell organelles , proteins , viruses, etc.

The analytical ultracentrifuge was developed by The Svedberg in the mid-1920s . It is mainly used to determine sedimentation coefficients and molecular weights . In this way, it provides information about the shape, conformational changes, size distributions and interactions of macromolecules .

Structure and principle of operation

The UZ separates dissolved particles using strong centrifugal forces generated in a rotating rotor . The separation process depends on the sedimentation properties of the particles (see also sedimentation speed ). These are determined by various factors, but above all by the size, density and shape of the particles. During the UZ, extremely high centrifugal forces act on the particles. Therefore, very small particles can also be separated. The strong centrifugal forces are made possible by the special structure of the UZ. In contrast to other centrifuges, the rotor is located in a vacuum chamber. Due to the vacuum , the rotor no longer experiences air resistance while it is running and can therefore achieve the very high accelerations and speeds. The heat actually generated by the rotor movement is prevented by cooling the vacuum chamber. The temperature of the vacuum chamber can be freely adjusted and thus optimally adapted to the often sensitive sample.

In the preparative as well as the analytical UZ, special types of rotors are used. The analytical UZ is also equipped with an optical measuring system.

Preparative ultracentrifugation

The preparative UZ is used to separate particles based on their size (simple and differential pelleting , zonal sedimentation) or their density ( isopycnic density gradient centrifugation ). UZs used for preparative purposes achieve the greatest accelerations and the highest speeds, up to 10 ⁶ g and up to 150,000 revolutions per minute (rpm).

Two types of devices are used in the preparative UZ: the large, floor-standing, ultracentrifuges (Fig. 1) and table-top ultracentrifuges (Fig. 2). Because of their smaller rotors, tabletop ultracentrifuges reach the highest speeds. That is why they are used to purify particularly small particles, such as ribosomal subunits. If larger sample volumes are involved, however, ultracentrifuges are used (table-top ultracentrifuge: up to approx. 195 ml; ultracentrifuge: up to approx. 560 ml). These achieve accelerations of up to 8 × 10 ⁵ g and speeds of 100,000 rpm.

A special type of table top ultracentrifuge is the so-called airfuge. Its rotor is driven by compressed air and can reach speeds of up to 110,000 rpm within 30 seconds. The Airfuge is therefore often used when particularly fast processing of the sample particles is necessary, e.g. B. if the sample is an unstable receptor- ligand complex.

Rotors

It is crucial for the successful outcome of an experiment that all materials used at UZ are optimally matched to one another and to the requirements of the sample. The choice of rotor is of central importance here. The best possible result is achieved when the rotor cleans or concentrates the particles in the sample with maximum efficiency. Often only its maximum speed is viewed as a measure of the efficiency of a rotor. In fact, its performance is determined by several factors. Three key factors are: 1) the angle at which the sample tubes are oriented, 2) the separation distance, and 3) the separation time. A simple measure of the overall efficiency of a rotor that takes all of these factors into account is what is known as the k-factor. This results as follows:

${\ displaystyle k = {\ frac {2.53 \ times 10 ^ {5} \ times ln (r_ {max} / r_ {min})} {(rpm / 1000) ^ {2}}}}$

The smaller the k-factor, the greater the efficiency of the rotor.

Figure 3 shows sample images of the various rotor types and how the samples are aligned in them.

There are three general types of rotors: fixed-angle rotors, swing-bucket rotors and vertical rotors (Fig. 3). The most noticeable difference between them is the angle at which the sample tubes are aligned with the axis of rotation of the rotor while the centrifuge is running and at rest. With fixed-angle rotors the sample tubes are inclined (between 20 ° and 45 °), with swing-bucket rotors the samples are horizontal, and with vertical rotors they are vertical. The orientation angle has a decisive influence on the creation of wall effects, which have a disruptive effect on the formation of particle bands.

Furthermore, the orientation angle determines the separation distance. This is the distance that the sample particles have to travel in the sample tube in order to pellet on the tube wall. The separation distance can be calculated from the difference between r _max (the distance between the rotor axis and the bottom of the tube) and r _min (the distance between the rotor axis and the beginning of the tube) (Fig. 3). Separation distances of the same rotors result from the diameter of the sample tube and the angle of orientation of the tube during the centrifuge run.

The maximum speed of a rotor and its r _max determine the maximum acceleration force of the rotor. Another important parameter is r _min , which determines the centrifugal force on particles in the uppermost part of the sample tube.

Special test tubes are used in practice to reduce the k-factor of a rotor and thus increase its efficiency. These tubes have a smaller volume, which reduces the separation distance. They are closed with a spacer. As a result, r _max remains unchanged and thus also the maximum acceleration of the rotor.

The right choice of rotor therefore depends on several factors. However, it depends first and foremost on the planned application, the nature and volume of the sample and the separation process required. In general, fixed-angle rotors are used for effective pelleting and isopycnic density centrifugation of macromolecules. Swing-bucket rotors are mainly used for isopycnic density centrifugation of cells and cell organelles as well as for zonal sedimentation. Vertical rotors are mainly used in density gradient centrifugation without pellet formation (Table 1).

Special rotor shapes

In addition to the three rotor types mentioned above, there are also some special forms of the vertical rotor. The zonal rotor and the flow-through rotor are of particular importance here (Fig. 4). These were specially developed for large sample volumes. They are used to separate larger sample particles such as bacteria, cells, cell organelles or viruses from tissue homogenates, but are also becoming increasingly important in the purification of nanoparticles. These rotors have an important application in the commercial manufacture of vaccines.

Figure 4 shows sample images of zonal and flow rotors.

The large capacity of these rotors (50-100 times higher than that of a conventional swing-bucket rotor) is achieved by dispensing with the use of individual sample vessels. Instead, the sample is placed directly in the mostly cylindrical rotor space. Large sample volumes can be separated in zonal rotors using density gradient centrifugation. To do this, the gradient medium is placed directly in the rotor space and then covered with the sample. Flow-through rotors are also equipped with a pump system that enables the sample solution to flow continuously through the rotor space during the centrifugation run. This enables sample throughputs of up to nine liters per hour to be achieved. These rotors are therefore particularly suitable for the sedimentation or concentration of large sample particles (> 50 S ) from very liquid sample solutions. Through a specially adapted loading and unloading process, flow-through rotors can also be used for density gradient centrifugation.

A third special form are the almost vertical rotors. The sample tubes are only slightly swiveled out here, between approximately 7.5 ° and 9 °. The shortened separation distance, in contrast to normal vertical rotors, reduces the separation or centrifugation time. The slight inclination prevents the solution and any gradient bands it may contain from coming into contact with the pellet at the end of the centrifugation run.

Easy pelletizing

Simple pelleting is one of the simplest and most widely used centrifugation techniques. It is typically used as part of a process for harvesting cells or for isolating precipitated material. Pelleting (sedimentation) refers to the separation of particulate and non-particulate material. The particulate material collects at the bottom of the sample tube during the centrifugation run and forms a solid pellet (sediment) there after a corresponding centrifugation time. Simple pelleting usually represents an early step in the complex and multi-stage processes for the purification of particles. The supernatant or the pellet is then discarded or processed further.

During the purification of proteins (main article protein purification ), for example, the protein to be examined is first z. B. overexpressed in bacteria that are cultivated in a nutrient solution. A large-volume centrifuge is used to "harvest" the bacteria from the solution. The bacteria form a pellet at the bottom of the tube. After discarding the supernatant nutrient solution, the pellet is processed further. When extracellular vesicles (EVs), for example exosomes , are purified , however, the pellet is discarded and the EVs are obtained from the supernatant. In the subsequent purification steps of both examples, further centrifugation methods are usually used, such as differential and density gradient centrifugation.

Differential pelleting

In differential pelleting (also called differential centrifugation ), several individual centrifugation runs are strung together. This method is often used for the separation of subcellular fractions (e.g. cell organelles, the main article of cell fractionation ) . First, a tissue or a bacterial suspension is homogenized by grinding or smashing, for example using a French press (main article French press ), and then floated with a physiological buffer solution. This homogenate is then subjected to successive centrifugation steps with increasing acceleration. After each centrifugation step, the supernatant is carefully separated from the pellet and subjected to another centrifugation run. With increasing acceleration, smaller and smaller particles can be pelletized. In the procedure usually carried out to purify cell organelles, the sediment would contain the cell nuclei as well as parts of the plasma membrane and whole, non-fragmented cells after the first run (10 min, approx. 600 g ) . After further centrifugation steps with the respective supernatant, the pellet of the fourth run (60 min, 10 ⁵g ) would contain the very small, soluble components of the cytoplasm such as enzymes , lipids and substances of low molecular weight such as salts and sugars. The cell fractions obtained in this way always contain several types of particles. These are often purified from the individual cell fractions by subsequent density gradient centrifugation.

Density gradient centrifugation

The density gradient is applied, for example, for further purification and separation of particles fractions from differential centrifugation. In contrast to this process, a solvent with a density gradient is used here. This allows fractions of macromolecules to be separated better. A substance (e.g. sucrose ) is added to the solvent , the concentration of which changes relative to the distance to the axis of rotation. This results in a location-dependent, varying density in the sample tube, a density gradient that acts on the macromolecules to be fractionated during the centrifugation run. This results in clearly separated bands with fractions of the particles that can be used for further preparative or analytical purposes. The most important types of centrifugation in the density gradient are zone sedimentation and isopycnic centrifugation.

Zone sedimentation

Zone sedimentation is used to separate particles according to their size, for example cell types, cell organelles but also proteins. The formation of the bands in this process depends on the duration of the centrifugation run, which is interrupted before a state of equilibrium between the solvent and the sample particles is established.

In the case of zone sedimentation, the gradient is covered with the sample to be separated. The density of the sample suspension is lower than that of the gradient, so that bands (zones) of different particles migrate with relatively good separation during the subsequent centrifugation. After a suitable sedimentation time, the bands can be harvested, e.g. B. by pipetting off the supernatant or by removing the band using a cannula. Flat gradients are mostly used for zone sedimentation; H. there is only a slight difference in density between the top and bottom of the tube.

The density gradient is important in forming or maintaining the bands for the following reasons. The centrifugal acceleration increases proportionally with the distance to the axis of rotation. That is, the sedimentation velocity acting on the particles increases towards the bottom of the centrifugation tube. Without a gradient, this leads to the particle bands diverging and overlapping. The gradient counteracts the increase in sedimentation speed in two ways. First, the density and therefore viscosity of the gradient also increases with the distance from the axis of rotation. The resulting increase in the coefficient of friction counteracts the sedimentation speed of the particles. Second, with increasing density in the gradient, the buoyancy that acts on the particles also increases, which also counteracts the increase in sedimentation speed.

Swing-bucket rotors or vertical rotors are usually used for this type of centrifugation (Fig. 3). Fixed-angle rotors are less suitable because, because of the collision of macromolecules with the wall of the centrifuge tube, bands that migrate undisturbed can hardly develop.

A typical application example is the separation of ribosomal subunits in a sucrose density gradient. For the separation of different cell types, e.g. B. Lymphocytes or cell organelles are mainly used Ficoll or Percoll gradients.

Isopycnic centrifugation

In isopycnic centrifugation, the separation of particles is based on their density. It is centrifuged until a sedimentation equilibrium has been established for the particles to be separated. The particle types have then formed bands at positions in the gradient that correspond to their buoyant density. The particles would not migrate further in the medium even with longer centrifugation times.

Self-forming and pre-formed gradients are used for isopycnic separation of macromolecules. Gradients for larger particles such as cells or organelles are usually preformed, that is, they are already in sedimentation equilibrium. This method can be used, for example, to separate particles in a homogenate. A mostly linear density gradient , e.g. B. a sucrose solution is given and overlaid with the sample solution.

Self-forming gradients are usually used to separate macromolecules and other small particles. The gradient is here during the centrifuge run with the onset of sedimentation of the gradient medium, z. B. concentrated saline solution formed. This type of isopycnic centrifugation is used, for example, to purify DNA . It delivers highly pure plasmid DNA. In the cesium chloride gradient, nucleic acids are separated based on their density. In the presence of ethidium bromide, chromosomal DNA can be separated from plasmid DNA. Ethidium bromide intercalates between strands of DNA, but is much more strongly attached to the plasmid DNA. This gives it a higher density and forms a band that is further away from the axis of rotation than that of the chromosomal DNA.

In isopycnic centrifugation, the gradient medium is overlaid with the sample, mixed, or the sample is introduced at the bottom of the gradient (flotation separation). The latter method is used for the separation of different types of lipoproteins as well as for the subfractionation of different membrane types.

The choice of rotor depends on the type of sample. For larger particles, the best results are achieved with swing-bucket rotors. Fixed-angle rotors are particularly suitable for separating macromolecules because they achieve the best resolution.

Table 1: Rotor types and their application
Particle	solvent	Procedure	Rotor type
DNA, RNA, proteins, macromolecules	Salts (CsCl, Cs ₂ SO ₄ , KI, ..)	isopycnic centrifugation	Fixed-angle rotors
Organelles, membranes	Sucrose, Optiprep, Nycodenz	isopycnic centrifugation	Swing-out rotors
Cell type separation	Percoll + osmotic buffer, Nycodenz, Optiprep	Zonal sedimentation	Swing-out rotors, vertical rotors

Analytical ultracentrifugation

With analytical ultracentrifugation , the movement of the analytes in the gravitational field is recorded online spectroscopically . There are two basic experiments to be distinguished. The sedimentation run provides the sedimentation coefficient . In the equilibrium run, there is a static concentration equilibrium , as in the hydrostatic equilibrium , from which the molar mass of the macromolecule can be determined with high accuracy, e.g. B. by density gradient centrifugation .

Examples of its use in biochemistry

for the isolation of lipoproteins (density gradient ultracentrifugation)
for concentrating proteins with a high molar mass , such as microsomes (differential ultracentrifugation)
As a cleaning step for the isolation of viruses and virus-like particles that are necessary for research or as starting materials ( antigens ) for the production of diagnostic tests (density gradient ultracentrifugation)
Determination of sedimentation coefficients and molecular weights of macromolecules

Individual evidence

↑ ^a ^b ^c ^d ^e ^f ^g ^h Gerold Adam, Peter Läuger, Günther Stark: Physical chemistry and biophysics (= Springer textbook ). Springer Berlin Heidelberg, Berlin, Heidelberg 2009, ISBN 978-3-642-00423-0 , doi : 10.1007 / 978-3-642-00424-7 ( springer.com ).
↑ ^a ^b ^c ^d ^e ^f Beckman Coulter Ultracentrifuge Product Catalog_CENT-1088CAT08.15-A
↑ Benjamin Rivnay, Henry Metzger: Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E . In: Analytical Biochemistry . tape 130 , no. 2 , April 15, 1983, ISSN 0003-2697 , pp. 514-520 , doi : 10.1016 / 0003-2697 (83) 90626-7 ( sciencedirect.com [accessed May 30, 2020]).
^ ^A ^b ^c ^d ^e D Rickwood: Preparative centrifugation: A practical approach . In: Biochemical Education . IRL Press, 1992.
↑ ^a ^b ^c ^d ^e ^f David Rickwood: Centrifugation Techniques . In: Encyclopedia of Life Sciences . John Wiley & Sons, Ltd, Chichester, UK 2001, ISBN 978-0-470-01617-6 , pp. a0002704 , doi : 10.1038 / npg.els.0002704 ( wiley.com ).
↑ ^a ^b From omniadm: The right rotor selection for optimal centrifugation | OMNILAB blog. February 26, 2020, accessed on May 30, 2020 (German).
↑ ^a ^b Claudia Simone Plüisch, Brigitte Bössenecker, Lukas Dobler, Alexander Wittemann: Zonal rotor centrifugation revisited: new horizons in sorting nanoparticles . In: RSC Advances . tape 9 , no. 47 , 29 August 2019, ISSN 2046-2069 , p. 27549–27559 , doi : 10.1039 / C9RA05140F ( rsc.org [accessed May 30, 2020]).
^ CB Reimer, RS Baker, RM van Frank, TE Newlin, GB Cline, NG Anderson: Purification of large quantities of influenza virus by density gradient centrifugation. Ed .: Journal of Virology. 1st edition. No. 6 , 1967, p. 1207-1216 .
↑ ^a ^b Beckman Coulter JCF-Z Zonal and Continuous Flow Rotor Manual
↑ Clotilde Théry, Sebastian Amigorena, Graça Raposo, Aled Clayton: Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids . In: Current Protocols in Cell Biology . tape 30 , no. 1 , March 2006, p. 3.22.1–3.22.29 , doi : 10.1002 / 0471143030.cb0322s30 ( wiley.com [accessed May 30, 2020]).
↑ Anurag Purushothaman: Exosomes from Cell Culture-Conditioned Medium: Isolation by Ultracentrifugation and Characterization . In: The Extracellular Matrix: Methods and Protocols (= Methods in Molecular Biology ). Springer, New York, NY 2019, ISBN 978-1-4939-9133-4 , pp. 233-244 , doi : 10.1007 / 978-1-4939-9133-4_19 .
↑ Susanne J. Bauman, Meta J. Kuehn: Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response . In: Microbes and Infection . tape 8 , no. 9-10 , August 2006, pp. 2400–2408 , doi : 10.1016 / j.micinf.2006.05.001 , PMID 16807039 , PMC 3525494 (free full text) - ( elsevier.com ).
^ ^A ^b John Graham: Preparation of Crude Subcellular Fractions by Differential Centrifugation . In: The Scientific World JOURNAL . tape 2 , 2002, ISSN 1537-744X , p. 1638–1642 , doi : 10.1100 / tsw.2002.851 , PMID 12806153 , PMC 6009713 (free full text) - ( hindawi.com ).
↑ SJ Garger, OM Griffith, LK Grill: Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient . In: Biochemical and Biophysical Research Communications . tape 117 , no. 3 , December 28, 1983, ISSN 0006-291X , pp. 835-842 , doi : 10.1016 / 0006-291X (83) 91672-8 ( sciencedirect.com [accessed May 30, 2020]).
↑ https://www.axis-shield-density-gradient-media.com/Isolation%20of%20cells.pdf
↑ https://axis-shield-density-gradient-media.com/Leaflet%20Nycodenz.pdf

[:0-1] ↑ ^a ^b ^c ^d ^e ^f ^g ^h Gerold Adam, Peter Läuger, Günther Stark: Physical chemistry and biophysics (= Springer textbook ). Springer Berlin Heidelberg, Berlin, Heidelberg 2009, ISBN 978-3-642-00423-0 , doi : 10.1007 / 978-3-642-00424-7 ( springer.com ).

[:1-2] ↑ ^a ^b ^c ^d ^e ^f Beckman Coulter Ultracentrifuge Product Catalog_CENT-1088CAT08.15-A

[3] Benjamin Rivnay, Henry Metzger: Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E . In: Analytical Biochemistry . tape 130 , no. 2 , April 15, 1983, ISSN 0003-2697 , pp. 514-520 , doi : 10.1016 / 0003-2697 (83) 90626-7 ( sciencedirect.com [accessed May 30, 2020]).

[:2-4] A ^b ^c ^d ^e D Rickwood: Preparative centrifugation: A practical approach . In: Biochemical Education . IRL Press, 1992.

[:3-5] ↑ ^a ^b ^c ^d ^e ^f David Rickwood: Centrifugation Techniques . In: Encyclopedia of Life Sciences . John Wiley & Sons, Ltd, Chichester, UK 2001, ISBN 978-0-470-01617-6 , pp. a0002704 , doi : 10.1038 / npg.els.0002704 ( wiley.com ).

[:4-6] From omniadm: The right rotor selection for optimal centrifugation | OMNILAB blog. February 26, 2020, accessed on May 30, 2020 (German).

[:5-7] Claudia Simone Plüisch, Brigitte Bössenecker, Lukas Dobler, Alexander Wittemann: Zonal rotor centrifugation revisited: new horizons in sorting nanoparticles . In: RSC Advances . tape 9 , no. 47 , 29 August 2019, ISSN 2046-2069 , p. 27549–27559 , doi : 10.1039 / C9RA05140F ( rsc.org [accessed May 30, 2020]).

[8] CB Reimer, RS Baker, RM van Frank, TE Newlin, GB Cline, NG Anderson: Purification of large quantities of influenza virus by density gradient centrifugation. Ed .: Journal of Virology. 1st edition. No. 6 , 1967, p. 1207-1216 .

[:6-9] Beckman Coulter JCF-Z Zonal and Continuous Flow Rotor Manual

[10] Clotilde Théry, Sebastian Amigorena, Graça Raposo, Aled Clayton: Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids . In: Current Protocols in Cell Biology . tape 30 , no. 1 , March 2006, p. 3.22.1–3.22.29 , doi : 10.1002 / 0471143030.cb0322s30 ( wiley.com [accessed May 30, 2020]).

[11] Anurag Purushothaman: Exosomes from Cell Culture-Conditioned Medium: Isolation by Ultracentrifugation and Characterization . In: The Extracellular Matrix: Methods and Protocols (= Methods in Molecular Biology ). Springer, New York, NY 2019, ISBN 978-1-4939-9133-4 , pp. 233-244 , doi : 10.1007 / 978-1-4939-9133-4_19 .

[12] Susanne J. Bauman, Meta J. Kuehn: Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response . In: Microbes and Infection . tape 8 , no. 9-10 , August 2006, pp. 2400–2408 , doi : 10.1016 / j.micinf.2006.05.001 , PMID 16807039 , PMC 3525494 (free full text) - ( elsevier.com ).

[:7-13] A ^b John Graham: Preparation of Crude Subcellular Fractions by Differential Centrifugation . In: The Scientific World JOURNAL . tape 2 , 2002, ISSN 1537-744X , p. 1638–1642 , doi : 10.1100 / tsw.2002.851 , PMID 12806153 , PMC 6009713 (free full text) - ( hindawi.com ).

[14] SJ Garger, OM Griffith, LK Grill: Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient . In: Biochemical and Biophysical Research Communications . tape 117 , no. 3 , December 28, 1983, ISSN 0006-291X , pp. 835-842 , doi : 10.1016 / 0006-291X (83) 91672-8 ( sciencedirect.com [accessed May 30, 2020]).

[15] ttps://www.axis-shield-density-gradient-media.com/Isolation%20of%20cells.pdf

[16] ttps://axis-shield-density-gradient-media.com/Leaflet%20Nycodenz.pdf